The leading cause of death in Japan is malignant neoplasm, and among others, the mortality of lung cancer overtakes that of stomach cancer to rank the first in men, while ranking the third in women. The mortality of lung cancer tends to increase every year. Lung cancer is histopathologically classified into the following four main tissue types: lung squamous-cell carcinoma and small-cell lung carcinoma (SCLC) developing in the hilar area of the lung, and lung adenocarcinoma and large-cell lung carcinoma developing in the lung field.
In particular, small-cell lung carcinoma rapidly proliferates and causes remote metastasis in the early stage, and therefore, in many cases, this carcinoma is discovered to be advanced cancer, which has already metastasized systemically, even at the time of initial diagnosis. In regard to the cure rate of this type of cancer, the cure rate in patients with limited disease (LD) small-cell lung carcinoma in which the lesion is limited only to one side of the lung field is approximately 20%; however, inpatients with extensive disease (ED) small-cell lung carcinoma in which the lesion has metastasized to both lungs or to other organs, cure is said to be practically difficult.
Furthermore, since small-cell lung carcinoma is highly sensitive to anticancer drugs, chemotherapy is considered as the first choice of therapy. On the contrary, non-small-cell lung carcinoma (non-SCLC) shows a low response rate for chemotherapy, and thus surgical therapy is considered as the first choice of therapy.
Therefore, small-cell lung carcinoma is a cancer which particularly necessitates early discovery and treatment even among various types of lung cancers, and for that reason, differential diagnosis of small-cell lung carcinoma and non-small-cell lung carcinoma is extremely important for making decision on the therapeutic strategy.
One of the methods for detecting lung cancer is the sputum examination. However, although the sputum examination is suitable mainly for the examination of lung squamous-cell carcinoma, there is a problem that the positive rate against small-cell lung carcinoma is low. Roentgenography is also a method widely used in the discovery of lung cancer, but concerning the lung squamous-cell carcinoma or small-cell lung carcinoma developing in the hilar area of the lung, there is a problem that the shadow of the heart falls on the hilar area, so that it is very difficult to take images of the shadow of cancerous tissues. Further, with regard to small-cell lung carcinoma, it is considered that even if those patients showing abnormal shadow of the lung field are diagnosed using sputum cytodiagnosis, simple chest roentgenography, CT scan, bronchoscopy and the like, early discovery of this type of lung cancer is not easy.
In addition, some of the testing methods for diagnosing cancer such as, for example, exposure to radiation, biopsy and bronchoscopy, cause pain in the patients, and also require highly expensive equipments, expert technology, and the like.
Therefore, research is being conducted to find a tumor marker which makes it possible, through a more convenient blood test, to diagnose cancer with high efficiency while the cancer is curable. Today, 30 or more tumor markers are being utilized in the discovery and diagnosis of cancer diseases, indication for monitoring the course of disease, diagnosis of recurrence, and the like.
Since lung cancer is classified into a variety of tissue types, a tumor marker which is effective for the discovery or diagnosis of all types of lung cancer, is not reported yet. Therefore, at present, effective markers are selected and used in accordance with each tissue type of lung cancer.
For instance, carcinoembryonic antigen (CEA) or sialyl Lex-i antigen is mainly selected and used for lung adenocarcinoma, while squamous-cell carcinoma related antigen (SCC) is mainly selected and used for lung squamous-cell carcinoma, and so is neuron-specific enolase (NSE) or the like for small-cell lung carcinoma.
However, NSE is disadvantageous in that: (1) the positive rate against curable early cancer is low; (2) a transient increase in the measured values due to treatment is recognized; (3) the measured values are increased due to hemolysis upon blood collection; and (4) the difference between the measured values obtained from small-cell lung carcinoma patients and the measured values obtained from normal persons is small. Therefore, NSE could not be necessarily said to be an effective tumor marker for small-cell lung carcinoma.
Gastrin-releasing peptide (GRP) is a brain-gut peptide composed of 27 amino acids, which was isolated from porcine stomach tissues by McDonald et al. in 1978, and has a gastrin secretion promoting action. The existence of GRP in human has also been confirmed, and a gene encoding human GRP was also cloned in 1984.
Yamaguchi et al. at the National Cancer Center in Japan conducted an investigation on the biological characteristics of small-cell lung carcinoma, which is thought to be derived from neuroendocrine cells, and in the course of the investigation, they examined 15 or more kinds of brain-gut hormones, including adrenocorticotropic hormone (ACTH), calcitonin and the like, and found that GRP is actively secreted from cultured small-cell lung carcinoma cell lines at the highest frequency and highest concentration (Non-Patent Document 1). They also established a radioimmunoassay (RIA) combined with a method for concentrating GRP in blood, and revealed that patients with small-cell lung carcinoma would exhibit higher blood GRP concentration as compared with healthy persons. However, since GRP is rapidly digested in the blood, concentration thereof in blood is low, and since the assay mentioned above requires a complicated concentration process, clinical application of the assay is difficult.
It was revealed by researches conducted thereafter that three species of GRP precursors (ProGRP) are produced in various cells by alternative RNA splicing (Non-Patent Document 2). These three species of ProGRP have in common from amino acid 1 to amino acid 98 of the amino acid sequence, while the amino acid sequence varies among one another, from amino acid 99 and the rest, because of alternative RNA splicing. This common portion of the amino acid sequence consisting of amino acid 1 to amino acid 98 is shown in SEQ ID NO: 1. Hereinafter, in the invention, unless stated otherwise in particular, the number indication of amino acid residues in ProGRP, partial sequences thereof, digests and the like, is based on the number indication of the amino acid sequence of SEQ ID NO: 1.
The amino acid sequence consisting of amino acid 1 to amino acid 27 of the three species of ProGRP is identical with the amino acid sequence of mature GRP having gastrin secretion promoting activity. These three species precursors are all digested by hormone precursor cleavage enzymes, into mature type GRP having an amino acid sequence consisting of amino acid 1 to amino acid 27, and a C-terminal fragment (ProGRP-Cfrag) which is a digested product of ProGRP having an amino acid sequence consisting of amino acid 31 and the rest, and having no gastrin secretion promoting activity.
Holst et al. (Non-Patent Document 3) reported that according to a radioimmunoassay (RIA) method making use of an antiserum directed against a peptide having an amino acid sequence consisting of amino acid 42 to amino acid 53 (hereinafter, referred to as ProGRP (42-53)), the level of ProGRP or ProGRP-Cfrag in the blood plasma of patients with small-cell lung carcinoma was high. However, in this method, precipitation and extraction processes were required, and the sensitivity was not sufficient.
Miyake et al. noted that ProGRP is more stable in the blood than GRP, and that an amino acid sequence consisting of amino acid 31 to amino acid 98, which is a common portion in the three species of ProGRP, does not show homology with the amino acid sequences of other proteins, and thus they established a highly sensitive RIA method which does not require precipitation and extraction processes, using an antiserum with a high titer obtained by using a recombinant peptide formed from the same amino acid sequence (hereinafter, referred to as ProGRP (31-98)) as an antigen (Non-Patent Document 1). It was shown by this method that ProGRP serves as an excellent tumor marker in the same manner GRP does.
However, although this method is advantageous in the aspect of not requiring an extraction process, the measurement requires a period of 4 days, and the sensitivity is insufficient, being only 10 pM (77.3 pg of antigen/mL). Accordingly, the ProGRP level in the blood serum of a normal person cannot be measured, and this method is not satisfactory yet for clinical application.
Furthermore, since the RIA methods of Holst et al. and of Miyake et al. as described above are inhibition methods, measurement can be made if even a portion of a fragment of ProGRP has antigenicity. However, the sensitivity is lower than that of sandwich methods, and it is difficult to achieve clinical application of the ProGRP measuring methods where sensitivity enhancement is required. That is, in order to lead the detection of ProGRP to clinical application, it is essential to increase the sensitivity of detection, and particularly, an antibody that can be used in sandwich methods is needed.
Yamaguchi, Aoyagi et al. developed, for the purpose of achieving clinical application of ProGRP as a tumor marker for small-cell lung carcinoma, a convenient and highly sensitive reagent for ProGRP measurement which is based on the principle of enzyme-linked immunosorbent assay (ELISA) and makes use of a sandwich method (Patent Document 1). This method gives results in about 2 hours and has high sensitivity (2 pg/mL). Therefore, the method is at present widely used in clinical applications, and it is clear that ProGRP has higher sensitivity and specificity to small-cell lung carcinoma as compared with NSE.
It was also found using this measurement method that the serum ProGRP levels also increased in neuroendocrine tumors (thyroid medullary cancer, and the like) and cancers exhibiting neuroendocrine tumor-like characteristics (esophageal small-cell carcinoma, pancreatic small-cell carcinoma, prostate small-cell carcinoma, and the like) as well as in small-cell lung carcinoma. Thus, it is conceived that this method will be applied to early discovery of these tumors or monitoring of the treatment in the future.
However, although the stability of ProGRP in the blood is higher than that of GRP, more fluctuation in the measured values is recognized as compared with other common tumor markers. Therefore, in those methods using ProGRP as the object of detection, there is a restriction that the test sample for measurement must be frozen from immediately after the collection of blood to the time of measurement and stored (Non-Patent Document 4).
Aoyagi developed a sandwich measurement method making use of two or more species of antibodies that recognize the amino acid residue 40 to amino acid residue 75 of ProGRP or the amino acid residue 40 to amino acid residue 79 of ProGRP, which are both internal regions of ProGRP (31-98) (Patent Document 3). This method gives relatively stable results by measuring a test sample which has been stored at 4° C., and exhibits a detection sensitivity that is almost equal to that of the method described in Patent Document 2. However, this method requires, as shown in Example 4 of Patent Document 3, an amount of test sample that is more than that of common immunoassay methods, which use an amount of 100 μL.    Patent Document 1: Japanese Patent No. 3210994    Patent Document 2: Japanese Patent Application Laid-Open (JP-A) No. 6-98794    Patent Document 3: WO 2006/117994    Non-Patent Document 1: Cancer Research, Vol. 54, pp. 2136-2140 (1994)    Non-Patent Document 2: Spindel, et al., Mol. Endocrinol., Vol. 1, pp. 224-232 (1987)    Non-Patent Document 3: Holst, et al., J. Clin. Oncol., Vol. 7, 1831-1838 (1989)    Non-Patent Document 4: RinshoKensa, Vol. 39, pp. 981-986 (1995)